Standardization of a silver stain to reveal mesoscale myelin in histological preparations of the mammalian brain.

BACKGROUND: The brain is built of neurons supported by myelin, a fatty substance that improves cellular communication. Noninvasive magnetic resonance imaging (MRI) is now able to measure brain structure like myelin and requires histological validation.

NEW METHOD: Here we present work in small and large biomedical model mammals to standardize a silver impregnation method as a high-throughput histological myelin visualization procedure. Specifically, we built a new staining well plate to increase batch size, and then systematically varied the staining and clearing cycles to describe the staining response curve across taxa and conditions. We compared tissues fixed by immersion or perfusion, mounted versus free-floating, and cut as thicker or thinner slices, with two-weeks of post-fixation.

RESULTS: The staining response curves show optimal staining with a single exposure across taxa when incubation and clearing epochs are held to within 3 to 9minutes. We show that clearing was slower in mounted vs free-floating tissue, and that staining was faster and caused fracturing earlier in thinner sliced and smaller volumes of tissue.

COMPARISON WITH EXISTING METHODS: We developed a batch processing approach to increase throughput while ensuring reproducibility and demonstrate the optimal conditions for fine myelinated fiber morphology visualization with short cycles (<9minutes).

CONCLUSIONS: We present our optimized protocol to reveal mesoscale neuroanatomical myelin content in histology across mammals. This standard staining procedure will facilitate multiscale analyses of myelin content across development as well as in the presence of injury or disease.